The present invention relates in general to methods of determining or monitoring the weight of organic molecules. It relates in particular to time of flight (TOF) measurement methods using laser desorption ionization of molecules to be measured.
Time of flight methods of determining molecular weight are normally used for large molecules such as organic molecules (having a molecular weight greater than about 100,000 daltons). Such molecules are generally so heavy that well known deflection mass spectrometric methods are ineffective. In deflection mass spectrometry, ionized species are produced in a vacuum and passed through a magnetic field. The extent to which they are deflected by the magnetic field provides a measure of their weight. Large organic molecules may be sufficiently heavy in relationship to their charge (charge/mass ratio) that they are not readily deflected by a magnetic field.
In time of flight methods of mass spectrometry, charged (ionized) molecules are produced in a vacuum and accelerated by an electric field into a time of flight tube or drift tube. The velocity to which the molecules may be accelerated is proportional to the accelerating potential, proportional to the charge of the molecule, and inversely proportional to the square of the mass of the molecule. The charged molecules travel, i.e. "drift" down the TOF tube to a detector. The time taken for the molecules to travel down the tube may be interpreted as a measure of their molecular weight.
Laser desorption ionization mass monitoring is a TOF mass monitoring method wherein charged molecules of a species to be measured, or analyzed, are produced by laser irradiation (in a vacuum) of a crystalline host matrix including a small proportion, for example between about 1:1000 and 1:10,000 of the species. Irradiation with ultraviolet (UV) radiation is generally preferred. The host matrix is selected to optimally absorb and transfer the energy radiation to the analyte. The absorbed energy is transferred to the analyte which is ejected or desorbed from the matrix in the form of charged molecules (ions). The desorbed, charged molecules are then accelerated into a drift tube. The time of flight of the molecules through the tube is generally determined by detecting the irradiating pulse and using the detected signal to start a timing process. Charged molecules generated by the irradiating pulse are intercepted by a detector after they have traversed the drift tube. A signal from the detector caused by the intercepted molecules is used to stop the timing mechanism thus establishing the time of flight. Molecular weight of a given analyte may be determined by relating the flight time required for molecules of the desorbed analyte to travel to the detector, to a linear function describing mass/charge ratio and flight time. The mass/charge ratio:flight time relationship is determined by calibrating the function using a standard of predetermined molecular weight.
Two classifications of LDIM instruments have been established, microprobe instruments and bulk analysis instruments.
In a microprobe instrument, laser irradiation is finely focused to a small spot on a foil containing the analyte. The laser radiation is in the form of a short pulse of very high power density. The power density is such that a small hole is produced in the foil. Analyte ions are desorbed from the foil and emerge from the hole. A commercially available LDIM microprobe instrument is described in Heinen, F., et al., Int. J. Mass Spec. Ion Physics, vol. 47, 1983, 19-22.
Bulk analysis instruments use moderately focussed beams, for example, beams focussed to a spot having an area greater than about 0.1 millimeters. The beams are incident on a surface including the analyte in a host matrix. The matrix and analyte are applied in the form of a thin crystallized layer or layers on a surface forming the tip of a sample probe. In the bulk analysis instrument, an area on the probe tip may be irradiated sequentially, with multiple laser pulses. This may be helpful, for example, in gathering statistical data on measurements.
In prior art ionization methods used in mass spectrometry, energetic or "hard" ionization processes, for example, using energy exchange within a gas discharge, may produce fragmentation of analyte molecules, i.e. the formation of metastable ions having a range of different weights.
In both microprobe and bulk LDIM methods the laser desorption ionization method produces what may be termed "soft ionization" of an analyte. Soft ionization provides that predominantly single charged unfragmented analyte ions are generated. Preferably, ions are desorbed by a laser pulse having an intensity just above that threshold intensity required to cause desorption. In a pulsed laser it is difficult to provide pulses of repeatable intensity particularly if a laser is operated intermittently. Further, thresholds may vary between matrix sample combinations. If a pulse has an intensity significantly greater than the desorption threshold, abducts may be formed by the addition of one or more matrix molecules to a sample. This causes a distribution of indicated molecular weights around a true value leading to measurement uncertainty or loss of mass resolution.
In LDIM, mass resolution is determined in terms of mass/difference in mass (m/.DELTA.m). This is a measurement of an instrument's ability to produce separate signals from ions (molecules) of similar mass. LDIM mass resolution is dependent upon the molecular weight of an analyte. Generally, mass resolution decreases as analyte molecular weight increases. A mechanism for this phenomenon is believed to be covalent abduct formation between analyte and matrix material.
Generally, molecular weight measurement accuracy reflects the uncertainty in assigning a molecular weight value to a given measurement of flight time. In addition, however, to uncertainties due to molecular weight of the analyte, a significant factor in limiting mass resolution is the uncertainty of the flight time measurement. Here, the primary limiting factor is that desorbed ions are released over a finite time interval which has some limit of reproducibility from one laser (desorbing pulse) to another. For example, if a molecular weight corresponds to a flight time of about twenty-six microseconds (26 .mu.sec), and if the desorbed analyte molecules are released over a period of about two-hundred nanoseconds (200 nsec) in a first pulse and 220 nsec in a subsequent pulse, then the maximum resolution from this uncertainty alone would be about one part in one thousand. The release time may be affected by the pulse width and spatial energy distribution, and repeatability of the laser radiation pulse causing the desorption. The release time may also be affected by the type and preparation of the sample on a probe tip. The flight time itself may be affected by vacuum conditions, for example by collisions between drifting species and residual gases in the vacuum enclosure.
A preferred detector for LDIM instruments is a microchannel plate (MCP) detector which accelerates an incident ion pulse through one or two plates comprising a matrix of microscopic tubes. As ions pass through the tubes, they generate ions by collision with tube walls. An MCP detector operates best when ions strike a multichannel plate at high velocity. Preferably, an accelerating potential of about minus five thousand volts should be applied to accelerate the ions. A microchannel plate, however, operates optimally when a potential not greater than one thousand volts is applied across it and is not limited by electron depletion.
Another source of uncertainty in LDIM measurements is the formation of ions of the same molecule having different charges or from the formation of clusters of two or more molecules each having one or more unit charges per molecules. These may be referred to as quasi molecular ions and will have different flight times in an LDIM instrument. As such, they may indicate that different molecules are present in a sample and thus lead to difficulty in assessing the purity of a sample.
Still another source of uncertainty in LDIM measurements may lie in the preparation of samples. It is important to lay down an even, reproducible layer of matrix and analyte on a sample probe tip. Usually a droplet of matrix/analyte solution is applied to a probe tip. The drop is then crystallized by applying a vacuum to the probe tip to remove volatile fluid components. If the droplet is irregular in shape then thickness and sample distribution in the crystallized layer can be nonhomogeneous leading to unreproducible measurement results. If vacuum application is not variable, highly lipophilic analyte/matrix mixtures will be difficult to crystalize since more volatile components of the mixture will cause less volatile components to bubble.
U.S. Pat. No. 5,045,694 discloses an electrospray method of applying matrix to a probe tip. Although this method appears to produce better matrix layers, it involves applying a potential of about five thousand volts to the probe tip during application of the layers. This makes the method somewhat hazardous and can lead to corona discharge between the probe tip and the spray apparatus which may damage the probe tip and spray apparatus.
In view of the foregoing it will be evident that although LDIM provides a potentially convenient method for monitoring molecular weight of large organic molecules, there is a need for improvement in many hardware aspects of the technology including sample preparation, delivery of laser pulses, ion optics, and detectors. There is also a need for improved signal processing technology to identify and eliminate uncertainties which may arise, particularly from the generation of quasi molecular ions.